Sequential vaccinations with divergent H1N1 influenza virus strains induce multi-H1 clade neutralizing antibodies in swine

Vaccines that protect against any H1N1 influenza A virus strain would be advantageous for use in pigs and humans. Here, we try to induce a pan-H1N1 antibody response in pigs by sequential vaccination with antigenically divergent H1N1 strains. Adjuvanted whole inactivated vaccines are given intramuscularly in various two- and three-dose regimens. Three doses of heterologous monovalent H1N1 vaccine result in seroprotective neutralizing antibodies against 71% of a diverse panel of human and swine H1 strains, detectable antibodies against 88% of strains, and sterile cross-clade immunity against two heterologous challenge strains. This strategy outperforms any two-dose regimen and is as good or better than giving three doses of matched trivalent vaccine. Neutralizing antibodies are H1-specific, and the second heterologous booster enhances reactivity with conserved epitopes in the HA head. We show that even the most traditional influenza vaccines can offer surprisingly broad protection if they are administered in an alternative way.

1.The title is quite hard to read in its current form, and in my opinion, doesn't do the paper justice.Perhaps 'Generation of multi-clade…", or the inclusion of "produced/induced by" instead of just "by"? 2. Line 59, page 3: suggest reconsidering the use of "on the other hand".

The authors present an alternative vaccination strategy, which their data supports.
However, prior immunity is likely to influence responses to conserved or semi-conserved head epitopes targeted by the three-dose heterologous vaccine regimen.Although the authors repeatedly mention the 2009 pandemic as an example of prior immunity to influenza being beneficial, it might be useful to address the suitability of the three-dose regimen to human vaccination in relation to prior immunity in the discussion.For example, some studies suggest prior immunity could be a hindrance via an original antigenic sin (OAS) mechanism (eg PNAS 2017 Nov 21;114(47):12578-12583).This could impact the effectiveness of the proposed three-dose heterologous vaccination regimen.
Overall, an excellent and intriguing study.

Craig Thompson, Warwick Medical School, University of Warwick, UK Reviewer #3 (Remarks to the Author):
This is an excellent manuscript by Van Reeth et al.By using a highly relevant and biologically significant animal model of influenza the manuscript offers additional insights into how vaccination regimes affect the breath of immune responses against influenza.Through a series of studies using 2 dose or 3 dose vaccination regimes using UV-inactivated/adjuvanted vaccine candidates against H1 subtype swine influenza strains.The data is consistent with what the investigators statement that "Three doses of heterologous monovalent H1N1 vaccine result in seroprotective neutralizing antibodies against 71% of a diverse panel of human and swine H1 strains, detectable antibodies 31 against 88% of strains, and sterile cross-clade immunity against two heterologous challenge strains."However, it is less clear that (the three dose heterologous strategy) "...is as good or better than giving three doses of matched trivalent vaccine".The reason for the latter statement to be less convincing comes from the lack of details on the amount of antigen in the TIV formulation; if it is prepared as it is assumed, then the amount of each antigen in the TIV is ⅓ of the amount delivered for each antigen with each monovalent vaccine.Given the relatively minor differences between serological responses from pigs in the 3XTIV group versus the G10-ARM08-CA09 group, but higher ASC levels in the latter; is it possible that such differences are due to lower amount of each antigen/dose in the TIV formulation?I believe the authors need to address this confusion to strengthen the conclusions in the manuscript.Fig 5 and 6 are the core of the ms and highly significant to the ms.However, there aspects of those figures that perhaps the authors would like to discuss further: 1) the 3XTIV group show less variations among individuals than the G10-ARM08-CA09 group, making the former a more reliable approach to vaccination against H1 viruses, 2) Although not considered to be neutralizing, anti-NP antibodies have been shown to confer some protection against influenza, anti-NP ELISA titers could add to the overall discussion and to establish whether those can be linked to protection after challenge.We want to thank you for your constructive criticism and for giving us the opportunity to submit a revised manuscript.Below are the responses to each of your comments, in blue.We feel that the manuscript has been substantially improved in the revision process.Many thanks for your time and effort.

Reviewer #1
In the manuscript by Van Reeth et al. the authors performed a comprehensive evaluation of homologous and heterologous prime-boost strategies of whole virus inactivated vaccines for swine influenza.The results show that broadly reactive and cross-protective antibodies against diverse IAV-S H1 viruses are obtained only after 3 immunizations with monovalent WIV vaccine candidates.Although this is an important observation, it does not represent a completely novel finding in the influenza field as there are other studies demonstrating that heterologous prime boost regimens elicit broader cross-reactivity and protection against influenza viruses.Because of the complexity of the experimental design, the manuscript and figures are difficult to follow.Below I provide some specific comments that may help the authors to improve the manuscript: Thank you for these helpful comments.Please find the response to each of them below.
(1) Antibody titers should be presented in titers and not scores.The scoring system presented is confusing and the use of titer and score interchangeably in the text complicates interpretation of the results.
We have left out the scoring system throughout the revised manuscript and now only use antibody titers.The text, tables and figures have been revised accordingly, as explained under point 2 and 3.
(2) All antibody scores were determined with pooled serum samples.This may mask experimental variability which is often observed in immunization studies in swine.Each animal in the treatment groups should be tested, antibody titers presented and group geometric mean titers presented in the figures.
We agree that it is ideal to present antibody titers of individual animals.The use of pooled sera, however, may be a solution in case of large numbers of serum samples and/or multiple serological assays and test strains.This practice has been used before in vaccination studies with influenza and HIV, and in clinical studies with SARS-CoV2.In the present study, we had serum samples from 121 individual pigs at 4 or 7 different timepoints.We used 5 different serological assays.The 3 functional assays (HI, VN and NI), which are very labor intensive, were also performed against large panels of heterologous H1/N1 strains and of heterosubtypic strains.Only for this set of assays we used pooled sera per group.We apologize if this was unclear in the original manuscript.
Functional assays against the most important strains and antigens were performed with individual sera.These include assays against the 4 vaccine and both challenge strains, 5 mutant H1 strains and a mosaic H5/1 strain.The table below (Table 4 in the manuscript) summarizes the assays performed at each timepoint and whether individual or pooled sera were used.We have now also performed additional HI, VN and NI assays to compare antibody titers obtained with individual and pooled sera for the 6 most important strains at 14 days after the final vaccination.The differences between antibody titers of pooled sera and geometric mean titers of individual sera are shown in the file "Addendum for reviewers -Comparison Ab titers pooled versus individual sera".While pooled sera generally had higher titers, the differences were ≤2-fold for most samples (81% of samples in HI assay, 86% in VN, and 80% in NI).The comparative table could be included in the Supplementary tables if the reviewer wishes so.Results available for all groups, 2-dose groups only, 3-dose groups only (3) the graphical representation of the data needs to be improved.The table format with the scores and color scheme is very difficult to follow.Bar graphs showing individual animal Ab titers would likely be a better way of presenting the data.
The table below presents an overview of all data, how they were presented in the original manuscript and how we tried to improve this in the revised manuscript.Because of the multitude of individual antibody titers, we did not manage to present all of them in bar graphs.The most important results, however, are presented as bar graphs showing individual data.These include 1) numbers of ASC and VN and NI titers against the vaccine and challenge strains in experiment 2 (Fig. 3), 2) HI titers against H1 head mutants (Fig. 5), 3) virus titers post challenge (Fig. 6).Table 8 and 9: antibody titers expressed as scores New Supplementary Tables 8 and 9 Please note that for the sake of clarity, we did not add error bars to the bar graphs, but standard deviations can be found in the Source Data files.

Data
We have made a few other changes to clarify the experimental design and graphical representations for the reader: -Table 4. "Overview of serological assays, timepoints and use of individual or pooled sera" has been added to the manuscript.-Fig.3 includes a schematic of the prime-boost regimens in experiment 2.
-The same structure and listing of experimental groups are used to represent serology results in Table 2, Fig. 2 and Fig. 4.

Reviewer #2
Van Reeth et al is an interesting study with noteworthy results in which responses to different homologous and heterologous vaccine regimens are assessed.Of particular interest is the three-dose heterologous regimen which produces a broadly cross-reactive H1 subtype response mediated by HA head domain epitopes.This response is also protective.The study is rigorous with sound methodology; antibody and APC responses are assessed using multiple assays, in addition to challenge studies.
The study is significant to the field because (i) it demonstrates that epitopes in the HA head domain can induce broadly cross-reactive responses via vaccination, and (ii) presents an intriguing approach to vaccination, which could improve protection against influenza in swine and humans.
In addition to this, as the authors elude, the study raises many further questions, such as which HA head epitopes outside of the antigenic sites and stem mediate the broadly reactive response induced by the three dose vaccination regimen.
Overall the study is through and well-written.I have several minor comments, which I have outlined below.
Minor comments: (1) The title is quite hard to read in its current form, and in my opinion, doesn't do the paper justice.Perhaps 'Generation of multi-clade…", or the inclusion of "produced/induced by" instead of just "by"?
We agree with the reviewer.It was a challenge to find a suitable title of no more than 15 words.We have now changed the title into "Sequential vaccinations with divergent H1N1 influenza virus strains induce multi-H1 clade neutralizing antibodies in swine" (2) Line 59, page 3: suggest reconsidering the use of "on the other hand".
Has been replaced by "assess" (4) Line 145, page 7, use of "per se concordant" is confusing.I would suggest rephrasing.
This sentence has been rephrased as "Post-vaccination NI antibody titers followed similar kinetics as HI/VN titers, but they were higher and showed broader crossreactivity." (5) The authors present an alternative vaccination strategy, which their data supports.However, prior immunity is likely to influence responses to conserved or semi-conserved head epitopes targeted by the three-dose heterologous vaccine regimen.Although the authors repeatedly mention the 2009 pandemic as an example of prior immunity to influenza being beneficial, it might be useful to address the suitability of the three-dose regimen to human vaccination in relation to prior immunity in the discussion.For example, some studies suggest prior immunity could be a hindrance via an original antigenic sin (OAS) mechanism (eg PNAS 2017 Nov 21;114(47):12578-12583).This could impact the effectiveness of the proposed three-dose heterologous vaccination regimen.
Thank you for this valuable suggestion.In the revised manuscript we mention the issue of prior immunity / original antigenic sin (OAS) in the final paragraph of the discussion.We did not go into detail because of the complexity of OAS and the many unknowns, but we refer to some highly relevant papers (refs. 47-50).One of these is an excellent review article (Linderman et al., 2021) in which the paper mentioned by the reviewer (Zost et al.PNAS 2017) is also discussed.
The following text was changed in / added to the discussion (Lines 421-436): "Our results cannot be directly extrapolated to humans, in which preexisting immunity will shape the antibody response to subsequent influenza virus exposures.The phenomenon of immune imprinting or "original antigenic sin" is complex and incompletely understood [47,48].Depending on the context, it may interfere with or potentiate the response to subsequent influenza vaccinations [27,49,50].Anyhow, the vastly different immune histories in people of different ages may impact the design and implementation of new prime-boost strategies in humans.Furthermore, influenza vaccines and vaccine policies also differ in swine versus humans.The strains for use in human influenza vaccine production are selected each year and generally show minimal antigenic differences with the previous year's strain… Pigs are natural hosts for both influenza and coronaviruses and an excellent animal model to study the immune response to sequential vaccinations [20,52], as well as the effect of prior immunity [53]." Overall, an excellent and intriguing study.

Reviewer #3
This is an excellent manuscript by Van Reeth et al.By using a highly relevant and biologically significant animal model of influenza the manuscript offers additional insights into how vaccination regimes affect the breath of immune responses against influenza.Through a series of studies using 2 dose or 3 dose vaccination regimes using UVinactivated/adjuvanted vaccine candidates against H1 subtype swine influenza strains.
The data is consistent with what the investigators statement that "Three doses of heterologous monovalent H1N1 vaccine result in seroprotective neutralizing antibodies against 71% of a diverse panel of human and swine H1 strains, detectable antibodies 31 against 88% of strains, and sterile cross-clade immunity against two heterologous challenge strains." (1) However, it is less clear that (the three dose heterologous strategy) "...is as good or better than giving three doses of matched trivalent vaccine".The reason for the latter statement to be less convincing comes from the lack of details on the amount of antigen in the TIV formulation; if it is prepared as it is assumed, then the amount of each antigen in the TIV is ⅓ of the amount delivered for each antigen with each monovalent vaccine.Given the relatively minor differences between serological responses from pigs in the 3XTIV group versus the G10-ARM08-CA09 group, but higher ASC levels in the latter; is it possible that such differences are due to lower amount of each antigen/dose in the TIV formulation?I believe the authors need to address this confusion to strengthen the conclusions in the manuscript.
Thank you for pointing out this confusion.The trivalent vaccine (TIV) contains the same amount of each antigen / vaccine strain (256 HAU) as the monovalent vaccines.We have now clarified this and added more details on the amount of antigen to the methods section.
See Lines 504-508 (Methods): "Each 2 ml vaccine dose contained 256 hemagglutinating units (HAU) of a single vaccine strain, or of each of two or three different strains, diluted in PBS and … Thus, the total antigenic mass in the bivalent and trivalent formulations was 2-and 3-fold higher than in the monovalent vaccines." Lines 326-328 (Results): "Three doses of heterologous monovalent vaccine were as effective as three doses of matched TIV vaccine and better than two doses, while the total amount of antigen per dose was 3-fold lower." (2) Fig 5 and 6 are the core of the ms and highly significant to the ms.However, there aspects of those figures that perhaps the authors would like to discuss further: 1) the 3XTIV group show less variations among individuals than the G10-ARM08-CA09 group, making the former a more reliable approach to vaccination against H1 viruses, 2) Although not considered to be neutralizing, anti-NP antibodies have been shown to confer some protection against influenza, anti-NP

Page 4 ,
Fig 1 legend: "P sequence values are shown in the upper right triangle, antigenic units in the lower left triangle" This is not obvious to everyone.Please consider adding notations on the figure itself

Table 4 .
Overview of serological assays performed with time points and use of individual and/or pooled serum samples Time points in days post vaccination (dpv) 1, 2 or 3 vaccine strains HI/VN challenge strains NI vaccine strains NI challenge strains HI/VN/NI diverse H1/N1 strains HI/VN/NI other HA/NA subtypes HI H1 antigenic site mutants ELISA anti-H1 stalk ELISA anti-NP Individual samples tested, Pooled sample per group.

Fig. 5 :
Fig. 5: Geometric mean Ab titers per group shown as bars, individual titers as dots